This invention concerns techniques for preventing sample loss from containers open to atmosphere, especially loss of liquid trifluoroacetic acid (TFA) due apparently to anomalous surface properties which, under certain circumstances, cause the liquid TFA and any solids in solution therein to creep up the walls of a container and even right out of, and down the external walls of a container.
TFA has been found to creep in this way in partially filled tubes open to the atmosphere at temperatures of around 20xc2x0 C.
This can be highly undesirable, particularly when it is used for cleaving synthesised molecules from resin beads in solid phase synthesis of organic molecules. In this application TFA, usually mixed with an organic solvent such as dichloromethane, is added to resin beads in a tube or other container, for example a microtitre plate, (container) on which organic molecules have been synthesised. These molecules are all attached, at some point on each molecule, to the beads and the TFA breaks the bond, which attaches them to yield the required molecule in solution in the TFA/solvent mix. The next step in the process is usually evaporation of the TFA/solvent mix (TFA mix) to yield the newly synthesised compound. If some of the TFA mix creeps out of the sample container it will take some of the compound with it and that amount of compound is normally lost. As synthesising such compounds can be very expensive this is highly undesirable. In multi-sample holders, such as microtitre plates, compound escaping from one well can migrate into an adjacent well causing cross-contamination.
According to one aspect of the present invention, creep of TFA may be reduced by the addition of dichloromethane to a sample so that the TFA content of the liquid is reduced to 20% or less.
According to another aspect of the invention, creep of TFA may be reduced by the addition of 10% water or another liquid to the sample. In general creep will be reduced but the compound may not cleave from its resin in dilute solutions of TFA.
It is of course a prerequisite that any added liquids will not react with the compound.
Creep of TFA liquid seems only to occur on surfaces which are in contact with concentrated TFA vapour. This form of creep may well occur in other liquids when in contact with concentrated vapour of the same liquid, and according to another aspect of the invention dichloromethane or water or other liquids may be added to reduce such creep of other liquids.
Five methods have been devised for preventing or minimising the effect of this phenomenon. In all these methods it is preferable though not essential to have a dry inert gas eg dry nitrogen, rather than air, filling that part of the container not filled with TFA mix. This is because the strong affinity of TFA for the water in atmospheric air causes other problems.
In a first method, the top of the sample container is sealed as soon as the TFA mix has been added. The TFA vapour, which is heavier than air, evaporates to form a layer over the liquid surface but cannot fill the rest of the empty space above the liquid because it cannot displace the air or other gas in the sample container above the vapour. If the TFA mix only partially fills the container the liquid film on the sides of the container will not rise to the top of the container. The sample containers need to be unsealed in a centrifugal evaporator only after they are subjected to enough centrifugal force (in the region of a few hundred G) to prevent creep when the sealing caps or closures have been removed.
In a second method a container is employed in which the top is formed with a central tubular entrance in the form of an unspillable inkwell. The container is filled with TFA mix to a level below that of the lowest level of the central downward pointing tubular entrance of the container. TFA vapours will occupy the central entrance tube up to a level slightly above that of the lower end of the entrance tube, but can go no further if gas or air, displaced in the container by the TFA vapour, is trapped in the annular space around the tubular entrance tube, below the upper wall of the container surrounding inwardly directed the tube. There is therefore no continuous path in the inside surface of the container wall, in contact with TFA vapour, up which the TFA liquid mix can creep. This solution to the problem is more convenient in that there is no need to cap the container after filling it with resin beads, TFA mix or other chemicals, so that there is no cap to be removed when the samples are to be evaporated as when rotated in a vacuum chamber.
The ring shaped top can be formed as an integral part of the tube or well, or a cap fitted to a straight tube, or a small cap fitted to each of the openings in a microtitre plate. In screw top vials (eg see FIG. 9a) the ring can be formed by inserting a short tube and sealing it hermetically (FIG. 9b).
In a third method the container is filled with an inert gas having a higher density than TFA vapour (such as sulphur hexafluoride) shortly before, or shortly after, adding the TFA to the container. The heavy gas is not so readily displaced as air or nitrogen and will prevent or delay the TFA vapour filling the container which, in turn, will prevent the liquid TFA mix from reaching the top of the container or delay the process sufficiently to prevent sample loss or cross-contamination arising from creep of TFA mix.
In a fourth method, the TFA vapour is continuously removed and replaced with air or an inert gas such as nitrogen. In this arrangement a tube may be inserted into the container to a position at which its lower open end is just above the TFA mix, and the top end of the tube is connected to a pump or reduced pressure collecting device, so as to suck out the TFA vapour as soon as it reaches the level of the lower end of the tube. A gas such as nitrogen or air, is admitted to the top of the container to compensate for the loss of gas volume caused by the removal of the TFA mix vapour. Under these conditions the TFA mix liquid rises as far as the lower end of the tube but no further, and again a barrier exists, above that level, up which the TFA will not in general creep.
This method has the added advantage that it promotes rapid evaporation of the TFA mix and can be used both as a method of preventing TFA creep and of evaporating the TFA mix. If this method is used to prevent creep, the suction flow rate is kept to the minimum required to reduce evaporation during the time the TFA mix is required to be in contact with the resin. For evaporation after the required period, the flow rate can be increased to give rapid evaporation.
In essence therefore, according to the invention, the phenomenon of liquid creep up the wall of a vessel containing the liquid, is prevented by creating a continuous region on the inside surface of the vessel which cannot come into contact with the liquid vapour which exists above the surface of the liquid in the vessel.
In a fifth method measures are taken to ensure that the whole of the surface of the container is already coated in liquid TFA mix but of a mix not containing the solid material in solution in the sample. Because the surface above the TFA mix sample level is already coated in TFA mix there is no tendency for further TFA mix containing the sample to creep up the walls.
This situation can be created, for instance, by spraying pure TFA mix onto the surface of the container before or after the containers are filled with sample. If the sample needs to remain in contact for a considerable time before evaporation can take place it may be necessary to repeat the application of pure TFA mix so that the walls above the sample do not dry out and draw up the TFA mix sample.
In most procedures for solid phase synthesis the TFA mix is evaporated in a centrifugal evaporator after it has achieved the required cleavage of the compound from the resin. Once sufficient centrifugal force has been applied to the sample, the tendency of the TFA mix to creep up the walls of the container is suppressed. The requirement to prevent or delay creep, so that the sample does not migrate out of its container, is therefore during the period prior to centrifuging, and until sufficient centrifugal force has been applied to the sample to prevent creep. If no such precautions are taken, creep to the top of the container can occur in a few minutesxe2x80x94typically 10-15 minutes. Samples frequently have to be held for considerably longer periods before they can be evaporated, and in this period they are usually held outside a centrifugal evaporator.
If a heavy gas such as sulphur hexafluoride is used to delay creep it may be necessary to repeat the action of filling the space above the TFA mix with the heavy gas several times if there is an appreciable delay in applying centrifugal force to the sample after adding the TFA mix.
Sometimes the TFA mix must be left in contact with the sample for an hour or longer to allow the cleavage action to be completed, before evaporation can be started.
In accordance with another aspect of the invention it may be desirable to apply centrifugal force to a container filled or partially filled with a liquid sample such as TFA, without applying vacuum, until the sample is ready to be evaporated.
Alternately in accordance with another aspect of the invention the samples liable to creep may be stored in a chamber filled with a heavy gas, possibly under a positive pressure (ie greater than atmospheric pressure) so that the possibility of the gas being displaced by TFA mix vapour is reduced.
If creep is prevented by sealing the containers after filling with TFA mix, means is required for removing the sealing means when evaporation of the mix is required. The seals are preferably removed after the application of centrifugal force, but this will normally only occur after the samples are in a vacuum chamber and are being rotated at considerable speed. This situation makes it difficult to remove the seals, and seals which blow off can interfere with the rotating samples.
One possible method is to use sealing caps which can be pushed into the tops of the containers manually or by automatic mechanical means, and held in place by a friction fit sufficient to provide a good seal, but not enough to prevent the caps being pushed out of the containers by a pressure difference of a fraction of an atmosphere as can be caused by the application of a partial vacuum to the exterior of the tubes. The pressure difference arises because the containers are normally sealed under atmospheric pressure although a higher pressure could be used at a cost of increased complexity in the apparatus.
Caps to seal microtitre plates are commercially available either in strips or as individual caps or as complete blocks to cover all 96 wells in a 96 well microtitre plate. These could be retained by means of small straps so that they would fly out of the wells on application of a partial vacuum but be retained close to the wells. The caps may partially block the tops of the wells, but this will not interfere with evaporation.
Similar capping and strapping arrangements could be applied to tubes, and arrays of tubes.
According to another aspect of the invention, particularly applicable to microtitre plates or arrays of tubes, centrifugal force is used to remove the sealing caps. In this arrangement sealing caps, which match the arrangement of the wells in the microtitre plate, are secured to a rigid backing plate and pins or rods extend from the backing plate into holes in the microtitre plate (eg see FIG. 5a).
When the wells in the plate have been filled, the backing plate is fitted to the microtitre plate. The caps align with and seal the wells, and the pins project through and a short distance, typically about 5 mm, beyond the bottom of the microtitre plate.
The fit between the pins and the holes in the plate, or between the caps and the wells, or both, is such that the weight of the plate is insufficient alone to cause the plate to drop relative to the backing plate, when the plate is located on a horizontal surface and stood on the ends of the protruding pins.
After placement in a centrifugal evaporator and spun, centrifugal force added to the weight of the microtitre plate will release and break the seals, and cause the plate to slide down the pins thereby creating a gap around the top of each of the wells, which will allow evaporation to take place.
A similar mechanism can be applied to an individual tube, or to an array of tubes.